1. Field of the Invention
The present invention relates to a laser microscope irradiating a pump beam and a Stokes beam having different frequencies onto a sample, and detecting an anti-Stokes beam emitted from the sample, thereby obtaining an image.
2. Description of the Related Art
To date, in fields of research in medical science or biology, the study of cell functions has been actively carried out. In recent years, not only cell functions are studied, but also, the need to directly study the relationship between the function which is the subject and the protein molecular structure has increased.
A method of directly observing Raman scattering light called a molecular fingerprint is widely used. In particular, attention has focused on CARS (Coherent Anti-Stokes Raman Scattering) spectroscopy which can easily eliminate fluorescence from a sample. CARS spectroscopy irradiates a pump beam and a Stokes beam onto a sample, and detects an anti-Stokes beam (anti-Stokes Raman scattering light) emitted from the sample.
As shown in FIG. 5 and FIG. 6, Jpn. Pat. Appln. KOKOKU Publication No. 4-51784 has disclosed a laser measuring device 100. The laser measuring device 100 has, as a laser light source, a pulse YAG laser 102 for oscillating a laser beam having a wavelength of 1064 nm. In front of the laser 102, a second higher harmonic generator 104 for converting the wavelength of a laser beam to 532 nm and emitting a pump beam ω11 (wavelength λ11) is provided. On the optical path of the beam ω11, a beam splitter 106 is disposed so as to divide the beam ω11 in two directions. The beam ω11 entering the beam splitter 106 is divided into a reflecting beam ω11 and a transmitted beam ω11′.
A mirror 108 reflecting the reflecting beam ω11 is provided on the optical path of the reflecting beam ω11.
On the optical path of the transmitted beam ω11′, there is provided a dye laser 110 converting the beam ω11′ to a Stokes beam ω22 having a different frequency (wavelength of 607 nm) and emitting it. Further, on the optical path of the Stokes beam ω22, a knife edge 112 for blocking half of the transverse cross-sectional pattern of the Stokes beam ω22 and making it semicircular is disposed.
Further, a dichroic mirror 114 is provided at the position where the pump beam ω11 and the Stokes beam ω22 intersect on the optical path. The pump beam (reflecting beam) ω11 passes through the dichroic mirror 114, and the dichroic mirror 114 reflects the Stokes beam ω22, and makes them into one beam ω11, ω22.
On the optical path of the beam ω11, ω22, a lens 116 for condensing the beam ω11, ω22 at a predetermined distance is provided. Further, a sample M is disposed at this condensing position. The beam ω11, ω22 is irradiated onto the sample M, and an anti-Stokes beam ω33 is generated from the sample M. Note that, in addition to the anti-Stokes beam ω33, the pump beam ω11 and the Stokes beam ω22 are included in the beam passing through the sample M.
On the optical path of the beams ω11, ω22, ω33, a lens 118 for making the beams ω11, ω22, ω33 into parallel light is provided. Further, on the optical path of the beams ω11, ω22, ω33, there is provided a knife edge 120 blocking only the mixed portion of the pump beam ω11 and the Stokes beam ω22. On the optical path of the beams ω11, ω33, an appropriate wavelength selector 122 blocking only the pump beam ω11 is provided.
Further, on the optical path of the remaining anti-Stokes beam ω33, two reflecting mirrors 124 for reflecting the anti-Stokes beam ω33 are provided. A spectroscope 126 for dividing the anti-Stokes beam ω33 into a spectrum is also provided on the optical path of the beam ω33. Further, in front of the spectroscope 126, a detector 128 is provided, and the divided spectrum is detected.
Moreover, a mini computer 130 is connected to the detector 128. Further, an image displaying device 132 is connected to the mini computer 130. The spectrum detected at the detector 128 is converted to an electric signal by using the mini computer 130, and the signal is displayed on the image displaying device 132.
Adjustment for matching the phases of the beams ω11, ω22, ω33 is carried out by removing the knife edge 120 from the above-described structure and detecting the whole anti-Stokes beam ω33 contained in the transverse cross-sectional pattern of the pump beam ω11. After the adjustment is carried out, only the mixed portion of the pump beam ω11 and the Stokes beam ω22 are blocked by the knife edge 120. Further, the anti-Stokes beam ω33, whose spatial resolution generated from a minute intersection point in space is high, is easily obtained.
Accordingly, the laser measuring device 100 has means in which the Stokes beam ω22 (wavelength λ22) is mixed in one portion of the transverse cross-sectional pattern of the pump beam ω11 (wavelength λ11) and is made incident on a sample M. Further, at the exiting side, only the mixed portion of the pump beam ω11 and the Stokes beam ω22 is blocked, and there is provided means in which the anti-Stokes beam ω33 contained in the transverse cross-sectional pattern of the other pump beam ω11 is extracted.
Such a structure relates to precise adjustment of a minimum crossed axes angle method having a high spatial resolution. However, basically, it is difficult to remove the possibility that misalignment of the optical axes will occur. For example, even if an ideal adjustment is carried out, there is the possibility that misalignment will occur due to effects such as changes in the environmental temperature, vibration, or the like. Thus, even if the exiting orientations of the pump beam ω11 and the Stokes beam ω22 coincide, there is the possibility that misalignment will occur.
Accordingly, due to misalignment occurring, the strength of the anti-Stokes beam is weakened. Moreover, there is not only this, but also the possibility that the pump beam and the Stokes beam, which do not contribute to the generation of the anti-Stokes beam, will be detected as noise components. Namely, in detecting the anti-Stokes beam, there is the drawback that deterioration of the SN ratio is caused.
Further, in the prior art, when misalignment occurs, there are cases in which the pump beam and the Stokes beam, which do not contribute to emission of the anti-Stokes beam, are irradiated onto the sample. Therefore, although this suffices when the sample is a chemical substance, in the case of living cells or tissues, there is the possibility that these two beams will damage the sample.